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Lymphatic endothelial cells materials and methods

a technology of lymphatic endothelial cells and materials, applied in the field of lymphatic endothelial cell materials and methods, can solve the problems of insufficient methods for obtaining isolated lymphatic endothelial cells, the therapeutic and diagnostic implications of the dysfunction of these interactions remain elusive, and the inability to ascertain the mechanism of action of these important regulators of lymphatic endothelial cell function, etc., to achieve the effect of improving the ability to manipula

Inactive Publication Date: 2006-11-30
LUDWIG INST FOR CANCER RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a method for isolating lymphatic endothelial cells from a biological sample, which can be useful in medicine and molecular biology. This method involves contacting the sample with an antibody that recognizes lymphatic endothelial cells and binds to them, resulting in the isolation of the cells from other endothelial cells. The antibody can be a polyclonal or monoclonal antibody that recognizes the extracellular domain of VEGFR-3. The method can be used with various biological samples, such as mammalian organs or tissues, and can be performed using immunohistochemistry or other techniques. The isolated lymphatic endothelial cells can be used for various applications, such as research and treatment of lymphatic system disorders.

Problems solved by technology

However, the biochemical signaling pathways activated via VEGFR-3 are less well characterized than those of VEGFR-2, making it difficult to ascertain the mechanism of action of these important regulators of lymphatic endothelial cells function.
In the absence of such information, therapeutic and diagnostic implications of dysfunctions of these interactions remain elusive.
However, despite the availability of these markers, at present, there are no adequate methods of obtaining isolated lymphatic endothelial cells.

Method used

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  • Lymphatic endothelial cells materials and methods
  • Lymphatic endothelial cells materials and methods
  • Lymphatic endothelial cells materials and methods

Examples

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example 1

Materials and Methods

[0219] The present example provides details of materials and methods employed throughout the application and in the Examples presented herein below.

[0220] Antibodies and growth factors. The primary antibodies used in immunofluorescence were mouse mAbs against human CD31 (Dako), vWF (Dako) or VEGFR-3 (clones 9D9F9, 2E11D11 and 7B3F9; Jussila et at., Cancer Res. 58:1599-1604, 1998), rabbit antiserum against human LYVE-1 (Banerji et al., J. Biol. Chem., 144(4)789-801, 1999), affinity purified rabbit anti-human podoplanin (Breiteneder-Geleff et al., et al., Am. J. Path., 154(2) 385-394, 1999) or rabbit anti-human VEGF-C (882; Joukov et al., EMBO J., 15:290-298, 1996). Monoclonal antibody against proliferating cell nuclear antigen (clone PC10) was from Santa Cruz Biotechnology. FITC- or TRITC-conjugated goat anti-rabbit IgG, goat anti-mouse IgG and donkey anti-mouse IgG were obtained from Jackson Immunoresearch. The rabbit antiserum against human VEGFR-2 was a kind...

example 2

Human Dermal Microvascular Endothelial Cells Consist of Distinct Populations of Blood Vascular and Lymphatic Endothelial Cells

[0229] The functions of different VEGF receptors have been extensively studied in transfected cell lines, but the lack of an appropriate cellular background can compromise results obtained from such studies. Therefore, the inventors set out to separate microvascular endothelial cells into specific constituent populations of endothelial cells of one type which were substantially free of other types of endothelial cells. More particularly, the inventors generated populations of lymphatic endothelial cells that were substantially free of blood vascular endothelial cells and vice versa. In order to pursue this endeavor and to elucidate VEGFR-3 signaling pathways promoting endothelial cell survival, the inventors used primary endothelial cells, human dermal microvascular endothelial cells (HMVEC) and human umbilical vein endothelial cells (HUVEC). It was determin...

example 3

Analysis of VEGFR Specific Ligands Used for the Cell Survival Experiments

[0233] VEGF is an endothelial cell mitogen which has been also shown to protect endothelial cells from starvation and TNF-a induced apoptosis via activation of VEGFR-2 (Gerber et al., J. Biol Chem., 273:30336-30343, 1998; Spyridopoulos et al., J. Mol. Cell. Cardiol., 29:1321-1330, 1997). The abilities of the different VEGFRs to promote endothelial cell survival were compared by using VEGFR specific VEGFs. The specificities of the growth factors used were determined using a cell survival bioassay. For the bioassay, Ba / F3 pre-B cells were stably transfected with a chimeric receptor containing the extracellular domain of human VEGFR-1, VEGFR-2 or VEGFR-3 fused with the transmebrane and cytoplasmic domains of the mouse erythropoietin receptor.

[0234] As expected, only VEGF and PIGF were able to induce the survival of VEGFR-1 / EpoR cells (FIG. 1A). VEGF, VEGF-C, VEGF-D and orf viral NZ2 (ORFV2-VEGF) were able to sup...

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Abstract

The present invention is directed to methods and compositions for isolating lymphatic endothelial cells from a mixed population of cells. More particularly, the inventors have found that certain antibodies that recognize the extracellular domain of VEGFR-3 can be used to specifically isolated lymphatic endothelial cells substantially free of other contaminating non-lymphatic endothelial cells. Methods and compositions for producing such cells and using such cells are described.

Description

FIELD OF THE INVENTION [0001] The present invention relates generally to materials and methods relating to the isolation of endothelial cells and, cells isolated according to the present invention. More specifically, the present invention is directed to obtaining populations of isolated lymphatic endothelial cells. BACKGROUND OF THE INVENTION [0002] The lymphatic system is a complex structure organized in parallel fashion to the circulatory system. In contrast to the circulatory system, which utilizes the heart to pump blood throughout the body, the lymphatic system pumps lymph fluid using the inherent contractility of the lymphatic vessels. The lymphatic vessels are not interconnected in the same manner as the blood vessels, but rather form a set of coordinated structures including the initial lymphatic sinuses [Jeltsch et al., Science, 276:1423-1425 (1997); and Castenholz, A., in Olszewski, W. L. (ed.), Lymph Stasis: Pathophysiology, Diagnosis, and Treatment. CRC Press: Boca Raton...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K48/00A61K39/395G01N33/567C12N5/08C12N15/09G01N33/50A61K38/00A61P17/06A61P27/02A61P29/00A61P35/00C12N5/071C12N5/10G01N33/15G01N33/53G01N33/543G01N33/566G01N33/569
CPCC12N5/069G01N2333/71G01N33/56966C12N2501/165A61P7/10A61P17/06A61P27/02A61P29/00A61P35/00A61P43/00
Inventor ALITALO, KARIMAKINEN, TAIJA
Owner LUDWIG INST FOR CANCER RES
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